Dendritic spines are essential for integrating excitatory and inhibitory inputs in the central nervous system. The fine structure of the spines, with necks as small as 100 nm and heads as small as 400 nm, allow them to form ultra-structural compartments with localized control of signaling and biochemical microenvironment. The ultra-structure of spines does not readily allow for standard methods of characterization, such as confocal imaging and electrophysiology. Recent progress in two-photon and STED imaging has been essential to resolving structure and location, but obtaining insight to the biochemical environment remains elusive. Our goal is to use a novel Polymer-Free Nanosensors (PFN) combined with 2-photon laser scanning microscopy to characterize the biochemical environment of dendritic spines. Specifically, we propose assessing ion dynamics in the spines, starting with sodium and then easily extending the platform to other ions such as chloride, calcium, and potassium. Since loading nanosensors, even ones as small as 20 nm, through the necks of dendritic spines might prove difficult, we propose developing and characterizing a novel type of nanosensor: a polymer-less formulation that has the mechanical properties of an oil rather than a bead. This would allow us to load PFNs into even small structures, and then monitor fluorescence intensity as a real-time, reversible indicator of analyte concentration. Since the spine neck forms the barrier that diffusionally isolates the synapse, one predicts that these altered morphologies perturb the biochemical and electrical compartmentalization of the synapse and spine. Our proposal will determine the role of the spine neck in shaping synaptically-evoked signals and will set the foundation necessary for revealing the functional consequences of the morphological changes seen in disease states.